CN102105494A - Nanocomposite materials based on metallic nanoparticles stabilized with branched polysaccharides - Google Patents
Nanocomposite materials based on metallic nanoparticles stabilized with branched polysaccharides Download PDFInfo
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Abstract
The present invention provides nanocomposite systems made of metallic nanoparticles stabilized with branched cationic polysaccharides, in particular alditolic or aldonic mono- and oligo-saccharidic derivatives of chitosan, and their preparation obtainable with aqueous solutions of these polysaccharides in the presence or absence of reducing agents. The peculiar chemical and physical-chemical features of these polysaccharides allow to form metallic nanoparticles homogeneously dispersed in the polysaccharidic matrix and an effective stabilization thereof. The properties associated with the nanometric dimensions and the presence of biological signals on polymeric chains may be exploited in applications with antimicrobial activities and of molecular biosensors.
Description
Invention field
The present invention relates to the nano complexes material that forms by the metal nanoparticle that is stabilized in the side chain cationic polysaccharide matrix, and preparation and be applied to purposes in biomedicine, pharmacy and the field of food.
The state of the art
The preparation of metal nanoparticle is the research field that nanotechnology is shown great attention to; In fact, many metals has optical property, antimicrobial properties and the catalytic performance of relevant with nanoscale usually uniqueness.Particularly, metal has significant application interests such as for example broad-spectrum anti-microbial activity of silver.On the other hand, bacterium, fungi and virus infection are represented serious problems in many situations, and special needs are felt in the realization that therefore has a product of extensive antimicrobial spectrum.In fact, metal for example silver, gold, copper, zinc and the use of nickel in the antimicrobial material field has great effect to market, particularly treats the market of local organization infringement (topic tissue lesion), therefore, and for example, such as Johnson﹠amp; Johnson
And Convatec
Company sold medicine recently based on the antimicrobial properties of silver nano-grain.
Usually, obtain nano particle from metal salt solution in the presence of reductive agent and stablizer, stablizer is used for preventing nanoparticle aggregate.For stable nanoparticles, suitably the polymers soln of dilution is widely used, and the polymers soln of described suitable dilution allows to obtain the nano-complex system, and wherein the metallic particles result is homodisperse.For above-mentioned application, polyelectrolyte solution for example polyphosphate, polyacrylate, poly-(vinyl vitriol), poly-(allylamine) (Henglein, A., J.Phys.Chem.1993,97 (21), 5457-5471), poly-(ethyleneimine) (Kuo, P.L.; Chen, W.F.J.Phys.Chem.B 2003,107 (41), 11267-11272; People such as Dai (Nano Lett.2002,2 (5), 497-501)) be effective especially.
Usually, the needed component of formation that obtains metal nanoparticle is: proper metal salt, reductive agent and serve as the polymkeric substance of the stablizer that is used for colloidal suspension.
US 2007/00036031 people such as () Karandikar discloses the method that is used for forming silver nano-grain in the presence of solvent, stablizer, tensio-active agent, reductive agent and heating.In stablizer, polymkeric substance for example polyacrylamide and polysorbate20 have been mentioned.
Nano-complex system in the synthetic cationic polymers matrix has been described in patent WO2007/147094A2 (people such as Sambhy V.), it is characterized in that tertiary N atom (ternary nitrogen atom) or quaternary nitrogen atoms (quaternary nitrogen atom), wherein the polymkeric substance of stable nanoparticles is poly-(4-vinylpridine) derivative, and Silver monobromide is used to form these silver nano-grains.
Patent WO2007/017901A2 people such as () Omray P. has reported for example by using the prescription of polysorbate, propylene glycol and Tenox PG dispersive silver nano-grain.
Described in patent WO2007/096606A1 (people such as Crowther N.) and be used to discharge method of metal nanoparticles, this metal nanoparticle is to prepare in the presence of the stabilization of polymer that is used for antimicrobial application.The polymkeric substance of mentioning has: poly-(methacrylic acid), polyimide, poly-(vinyl alcohol) and multipolymer thereof.
Patent WO2007/001453 people such as () Yacaman M. has reported by using glycerine or ethylene glycol not only as reductive agent but also as the nanostructure solvent with by using poly-(V-Pyrol RC) coating-forming agent as nano particle, synthetic silver nano-grain in the presence of polyvalent alcohol and polymkeric substance.Notice that the chemical reduction mechanism of silver ions is based on the fracture of oxidation that is present in the hydroxyl in the polyvalent alcohol and C-C key.
For the application of biomedical sector, paid close attention to especially based on the system of polysaccharide, because these polymkeric substance are normally biocompatible, and therefore be suitable for relating to the application that directly contacts with biological tissue.In natural polysaccharide, be studied and one of the natural polysaccharide of commercial use is a chitosan morely.This is a kind of alkaline polysaccharide, has 50 to the molecular weight of 1500kDa, by by β 1 → 4 key and D-glucosamine (GlcNH from the residue N-ethanoyl-glucosamine unit bonded straight chain of the deacetylated distribution of incomplete chitin
2) the residue composition.This polysaccharide is soluble usually in neutrality or alkaline aqueous solution; In pH was equal to or less than 5 acidic solution, free amino was by protonated, thereby made polymkeric substance solvable.This polymkeric substance has been widely used in medical field, because it presents low immunogenicity, pathologic or infectious response (Suh Francis J.K., Matthew H.W.T.Biomaterials, 2000,21,2589-2598; People such as Miyazaki S., Chem.Pharm.Bull., 1981,29,3067-3069).Because the physical-chemical performance of chitosan is the high-cation electric density in acidic solution, its high working property energy and the ability that produces vesicular structure in the place of for example implantable cell for example, chitosan has all ideal features that are used as biomaterial.In fact, the many purposes of chitosan in biomedical and field of food are known.Because these performances, both also are used to stable nanoparticles chitosan and derivative thereof.
In patent WO2007/025917A1 (people such as Schmid H.), mentioned chitosan and chitosan derivatives and salt for example cm-chitosan, chitosan acetate and chitosan lactate in the presence of prepare silver nano-grain.
In patent US 2008/0147019 (Song Xuedong), add commercially available preformed metal nanoparticle to chitosan salt and chitosan derivatives, rather than have the side chain derivative of sugared characteristic (saccharidic nature); These nano particles need further oxidation so that anti-microbial activity to be provided, because silver ions (Ag
+) pointed out as unique antiseptic-germicide.
In patent WO2008/076339 (people such as Schauer C), commercially available metal nanoparticle is included in the material based on water-fast cross-linked chitosan.Netted chitosan is used to prepare multilayer film to support nano particle, and nano particle is used as staining agent and is used as the transmitter that detects the pollutent in the water.
Some nearest researchs have concentrated on the method for improving the biological effect that is used to improve chitosan.Particularly, most of effort all relates to increases the positively charged ion of polymkeric substance feature or changes its chemical feature and bioavailability by (biology) chemical modification.
Particularly, with glycosyl group carry out chitin modified, for example by utilizing the reductive amination reaction to insert the lactose unit, cause higher water-soluble chitosan derivative, as at US 4,424,346 (Hall, L.D. and Yalpani, mentioned in M.), and chitosan is to obtain the most useful performance with these deutero-forms to be used for as highly biocompatible biomaterial really.
General introduction
First purpose of the present invention is to obtain nano complexes material, wherein, metal nanoparticle be stabilized and be controlled in size system, and the performance of nano complexes material is particularly suitable for biomedical and optics (biosensor) field.
Another purpose is to utilize non-complex and chemical process that have superiority economically, and particularly can obtain such nano-complex by colloidal solution, and wherein metal nanoparticle is stablized by suitable polysaccharide soln.
Another purpose is by using the commercial polysaccharide that can obtain easily and making these polysaccharide avoid standing chemical operation and avoid the needs of the preparation manipulation of the complexity of these systems are improved these systems.
Another purpose is under the condition of big ionic strength required in neutral pH and biomedical applications, obtains completely soluble system in water system.
In order to achieve the above object, the inventor has developed the suitable soluble polysaccharide system based on the side chain cationic polysaccharide, and it allows to obtain metal nanoparticle stable, controlled in size system.These programs based in the existence of external reductive agent or not with the aqueous solution of aqueous polymers solution and metal-salt.This combination process can be carried out in the water system under the neutral pH expediently, and does not need heating, because it can at room temperature carry out easily.
Therefore, in first aspect, purpose of the present invention is provided by nano complexes material, and described nano complexes material is characterised in that it comprises polymeric matrix of being made up of the side chain cationic polysaccharide and the metal nanoparticle that is dispersed in this polymeric matrix.
The preferred cation polysaccharide is the side chain derivative of chitosan, and the D-glucosamine unit that wherein forms the chitosan straight chain connects (alditolic) or (aldonic) polyvalent alcohol residue of alditol of the sugar alcohol of being represented by general formula (I) that is same to each other or different to each other by means of the functional group-NH-on the carbon atom C2:
Wherein:
-R is-CH
2-or-CO-;
-R
1Be hydrogen, monose, oligosaccharides;
-R
2Be-OH or-NHCOCH
3
On the other hand, preferably silver, gold, platinum, palladium, copper, zinc or nickel and composition thereof of the metal of nano particle.
According to nano complexes material of the present invention can in the existence of external reductive agent or not, the aqueous solution with these polysaccharide and metal-salt under suitable condition prepares.
Therefore,, an object of the present invention is the preparation method of these nano complexes materials, may further comprise the steps according on the other hand:
A) preparation concentration is up to the aqueous solution of the side chain cationic polysaccharide of 2%w/v;
B) aqueous solution of the metal-salt of preparation concentration between 0.1mM and 20mM;
C) these metal salt solutions are added to polysaccharide soln and mixing until obtaining colloidal solution, wherein metal nanoparticle is a dispersive equably.
Reductive agent is randomly added to the colloidal solution that is obtained.
According to other aspect, the purposes of these nano complexes materials in biomedicine, pharmacy and field of food, for example as biocide, or the purposes in molecular biosensor is another object of the present invention.
The accompanying drawing summary
Fig. 1.The figure shows silver nano-grain with chitosan derivatives (the following Chitlac that also is called of lactose; CAS registration number 85941-43-1) stabilization in: the Chitlac polymer chain allows the coordination of metal nanoparticle and stablizes.The TEM image that obtains by Philips EM 208 transmission electron microscopes has confirmed that silver nano-grain (black particle) exactly is positioned on the polymer chain (grey branched structure).By using Chitlac (0.2%w/v) and 1mM AgNO
3Prepare sample (embodiment 7).
Fig. 2.The figure shows: (A) at the same terms (0.2%w/v polymkeric substance; 1mM AgNO
30.5mM xitix (C
6H
8O
6)) under, the UV-visible spectrum of the silver nano-grain that under the existence of chitosan (dotted line, embodiment 22) and Chitlac (solid line, embodiment 7), forms; (B) Chitlac and AgNO
3Concentration to the influence of UV-visible spectrum: 0.4%w/v Chitlac/1mM AgNO
3(solid line, embodiment 9); 0.2%w/v Chitlac/1mMAgNO
3(dotted line, embodiment 7); 0.1%w/v Chitlac/1mMAgNO
3(spaced points line
, embodiment 5); 0.1%w/v Chitlac/0.5mM AgNO
3(near dotted line
, embodiment 4); 0.2%w/v Chitlac/0.5mM AgNO
3(hyphen-dotted line
, embodiment 6); 0.4%w/v Chitlac/0.5mMAgNO
3(hyphen-point-dotted line
, embodiment 8).According to the stoichiometry of reaction, the AgNO in each sample
3/ C
6H
8O
6Ratio is 2.This measurement is to carry out under 25 ℃ temperature by the CaryE4UV-visible spectrophotometer.
Fig. 3.The figure shows (0.2%w/v polymkeric substance under the situation that does not have external reductive agent; 1mM HAuCl
4, embodiment 18), the UV-visible spectrum of the gold nano grain that in Chitlac, forms.Carrying out described in this measurement as previous Fig. 2.
Fig. 4.The figure shows and exist and do not exist under the situation of xitix, the Ag of the different silver concentrations of 0.2%w/v Chitlac/
nThe UV-visible spectrum of the sample of (silver nano-grain).Prepare sample according to the program described in following examples: embodiment 7 (1mM AgNO
3/ 0.5mM C
6H
8O
6, solid line), embodiment 10 (5mMAgNO
3, line
), embodiment 11 (10mMAgNO
3, line
), embodiment 25 (0.2%w/v Chitlac, 1mM AgNO
3, line---).Carrying out described in this measurement as previous Fig. 2.
Fig. 5.Under the situation that does not have external reductive agent, the silver nano-grain that in Chitlac, forms (0.4%w/v Chitlac, 14mMAgNO
3, embodiment 15) the TEM image.
Fig. 6.The figure shows under the situation that does not have external reductive agent, be dispersed in gold nano grain (0.2%w/v Chitlac, 1mM HAuCl among the Chitlac
4, embodiment 18) the TEM image.Carrying out described in this measurement as previous Fig. 1.
Fig. 7.The figure shows the gold nano grain (0.2%w/v Chitlac, the 1mM HAuCl that are dispersed among the Chitlac
4, embodiment 18) distribution of sizes.This measurement is by using Nanosight LM20 system to carry out.Particle size is 10 to the scope of 150nm, and most of particle has the size of about 50nm.
Fig. 8.The figure shows the copper nano particles (0.2%w/v Chitlac, the 1mM CuSO that are dispersed among the Chitlac
4, 0.2mM NaBH
4, embodiment 19) the TEM image.Carrying out described in this measurement as previous Fig. 1.
Fig. 9.The figure shows the silver nano-grain (0.2%w/v Chitlac, the 1mM AgNO that are dispersed among the Chitlac
3, 0.5mM C
6H
8O
6, embodiment 7) TEM image (A, B) and distribution of sizes (C).Carrying out described in this measurement as previous Fig. 1.
Figure 10.The figure shows: (A) intestinal bacteria ATCC 25922 is respectively at 20%Mueller-Hinton substratum (20%Mueller-Hinton; Contrast, trilateral) in or in the existence of 0.2%w/v Chitlac (circle, embodiment 1) down or at 0.2%w/v Chitlac+1mM AgNO
3+ 0.5mM C
6H
8O
6Bacterial growth kinetics under the existence of (square, embodiment 7).The data of being reported relate to have can correlated result three independent experiments; (B) intestinal bacteria ATCC25922 bacterium colony is at 0.2%w/v Chitlac+1mMAgNO
3+ 0.5mM C
6H
8O
6Counting in (solid line, embodiment 7).Dotted line is illustrated in the contrast that does not exist under the Chitlac-nAg.The result be at least 4 independent experiments mean value (± SD).
Figure 11.The figure shows intestinal bacteria ATCC 25922 at Mueller-Hinton substratum (20%Mueller-Hinton; The contrast, circle) in or at 0.2%w/vChitlac+1mM HAuCl
4Bacterial growth kinetics in (embodiment 18 for Chitlac-nAu, square).
Figure 12.The figure shows intestinal bacteria ATCC 25922 bacterium colonies in 20%Mueller-Hinton (contrast, circle) or the gold nano grain in being dispersed in Chitlac (0.2%w/vChitlac, 1mM HAuCl
4, embodiment 18) and counting under the existence of (square).CFU is the colony-forming unit of bacterium.
Figure 13.The figure shows and silver nano-grain (0.4%w/v Chitlac, 14mM AgNO
3, embodiment 15) the correlated 5%w/v concentration of Chitlac SERS spectrum (solid line) under Chitlac Raman spectrum (embodiment 1) (dotted line).
Figure 14.The figure shows of the influence of the methacrylic acid sample of coating to the LDH leakage of human osteosarcoma (MG63) clone.By both directly contact and carry out the cytotoxicity test with cell and material itself (MAcr nAg C) and with the liquid nutrient medium (" extract ") (MAcr nAg E) that keeps in touch the given time (24 hours with 72 hours) with this material.The control cells of in the adhesion (Ad) in the DMEM substratum completely, cultivating and with polystyrene dish (PS, negative control) and parallel the carrying out of corresponding group with cell that the urethane dish (PU is according to the positive control of ISO 10993-5) of ZnDBC handles and methacrylic materials that does not have nano particle (MAcr C, MAcr E).The TritonX-100 of 0.1% in the substratum is used as the positive control of extract.By after deducting contrast, calculate the per-cent of the LDH of release except that the activity amount in the substratum with total activity (substratum and cell lysates).Data are expressed as the mean value (having the SD scope) of four independent experiments.
Detailed Description Of The Invention
Definition
Colloidal solution (or colloid): its implication is a kind of system, and wherein, the particle with 1 to 1000nm size range is dispersed in the continuous solvent medium.
Nano-complex: the system that its implication is made up of the particulate (filler) that has nano-scale within macroscopic material (matrix). In description of the invention, term " nano-complex " refers to certain material, and wherein metal nanoparticle is dispersed in the polysaccharide matrix. Particularly, it refers to certain material, and wherein metal nanoparticle is that reduction by corresponding salt forms in the polysaccharide matrix basically, thereby major part all has zero charge, but does not get rid of the cluster that has on a small quantity ion characteristic (Ag for example4 +) existence. Therefore, except simple " nano-complex ", below also can refer to " metal-based nano compound " according to nano-complex of the present invention.
To understand better by the following detailed description and to comprise purpose and the advantage that is dispersed in nano complexes material metal nanoparticle, the object of the invention between side chain alkaline polysaccharide matrix, in the following detailed description, as nonrestrictive example, except the biological test of estimating its antibacterial activity, preparation also will be described according to some examples and the physical-chemical sign thereof of nano-complex of the present invention.
Describe
In the polymeric matrix that is made up of the side chain cationic polysaccharide, because the existence of uncle nitrogen-atoms and secondary nitrogen-atoms, the nano complexes material of the object of the invention comprises evenly and stably is dispersed in metal nanoparticle in such polymeric matrix.
For the purposes of the present invention, preferred cationic polysaccharide is side chain derivative sugar alcohol or alditol of shitosan, the D-Glucose amine unit that wherein forms the shitosan straight chain connects by means of the functional group-NH-on the carbon atom C2 and is same to each other or different to each other and by the monose of the previous general formula of reporting (I) expression or polyalcohol residue sugar alcohol or alditol of oligosaccharides, wherein R can be-CH2-or-CO-, and R1Can be hydrogen or monose or oligosaccharides, monose preferably be selected from the group that is made up of galactolipin, glucose, mannose, N-acetyl group gucosamine and GalNAc, and oligosaccharides preferably includes 2 glycoside units, and R2Can be-OH or-NHCOCH3 Below these derivatives also referred to as sugar alcohol or alditol, monose or the oligosaccharide derivative of shitosan, or referred to as monose or the oligosaccharide derivative of shitosan.
As representative, the D-Glucose amine unit that replaces with monose or oligosaccharides residue in the chitosan derivatives of the object of the invention can be represented that by general formula (II) wherein " n " refers to the sum of the D-Glucose amine that forms the shitosan straight chain:
Mentioned among the patent WO2007/025917A1 that formerly quotes (people such as Schmid H.) chitosan derivatives and salt for example CMC, chitosan acetate and chitosan lactate in the presence of prepare silver nano-grain. Be different from the above-mentioned patent of quoting, the derivatization in this case is to be undertaken by the structure of monose or oligosaccharides, and the structure of described monose or oligosaccharides is reacted by reductive amination, causes being connected to by means of secondary amino group the insertion of the side chain of polymer backbone. This allows i) amino still can be used for metal-complexing, and ii) total polysaccharide electric charge is not changed basically.
In fact, polymeric matrix is made up of the side chain derivative of shitosan, and the side chain derivative of described shitosan connects the monose of general formula (I) or derivative alditol or sugar alcohol of oligosaccharides by means of amino. The monose of these general formulas (I) or oligosaccharides preferably include 1 to 3 glycoside units, and according to preferred aspect, these are the oligosaccharides residues that comprise 2 to 3 glycoside units, and more preferably, according to R, R1And R2Different implications, these are selected from the group that is made up of following: the residue of lactose, cellobiose, cellotriose, maltose, maltotriose, chitobiose, chitotriose, mannobiose and corresponding glycuronic acid thereof. For the purposes of the present invention, the most preferred oligosaccharide derivative of shitosan is derivative (the following Chitlac that also is called with lactose; CAS registration number 85941-43-1).
In addition, for the purposes of the present invention, the chemical substitution value of amino of chitosan and these monose or oligosaccharides is at least 20%. Preferably, the substitution value of amino of chitosan and monose or oligosaccharides is 50% to 80% scope, and more preferably is 70%.
The mean molecule quantity (hereinafter referred to as MW) that can be used for the shitosan of the oligosaccharide derivative that obtains to mention can be up to 1500kDa, and preferably in the scope of 400kDa to 700kDa.
The metal nanoparticle that is attached in the polymeric matrix that is made up of these side chain cationic polysaccharide derivatives is preferably to be selected from following metal: silver, gold, platinum, palladium, copper, zinc, nickel and composition thereof. Be included in nano particle in the polymeric matrix that the oligosaccharide derivative by shitosan forms and have size from 5nm to the 150nm scope, and especially, the average metal nanoparticle size of control 30 and 50nm between. As will be from following detailed description of the present invention significantly, the essential feature of these nano particles is: these nano particle major parts all are the metals that exists with its reduction form, and do not get rid of residual by former molecular bunch the existence that has on a small quantity ion characteristic; And in the dispersion of these nano particles in the polysaccharide matrix/stable, relate to the oligosaccharides side chain near the amino of shitosan itself.
Be not subjected to the latter's restriction, preferred ratio between matrix polysaccharide and the metal is mentioned the nano-complex of colloidal solution form, although these metal-based nano composite materials can also be the film of dehydration or the form of powder and even can also be by dialysis so that residual counter ion is removed from the preparation of material itself. The aqueous colloidal solution form according to nano particle of the present invention in, the ratio of polysaccharide concentration (being expressed as %w/v) and initial metal salt concentrations (being expressed as molar concentration) is 0.0025 to 20, and is preferably 0.2.
Therefore, the silver amount that is expressed as mg that can be attached to every gram polysaccharide can be 3000mg/g to 0.3mg/g, and preferably 50mg silver is attached to every gram polysaccharide.
For the purpose of pursuing, the inventor has solved the aspect relevant with a kind of Preparation and characterization of the system based on polysaccharide, wherein, utilized the performance relevant with the nano metal size (antimicrobial properties of metal itself, optical property, catalytic performance and other properties) of the metal nanoparticle in the nano-complex that is included in the object of the invention. Hereinafter play-by-play these aspects.
The preparation of the oligosaccharide derivative of shitosan
The process of these oligosaccharide derivatives of preparation shitosan is at US 4,424,346 (Hall, L.D. and Yalpani, M.) known and description in, and be included under the existence of sodium cyanoborohydride, process shitosan and the solution of acetic acid (pH 4.5) in methyl alcohol with selected monose or oligosaccharides. Interaction between the aldehyde radical of the amino of shitosan and monose or oligosaccharides causes being called the formation of the unsettled intermediate of schiff bases. In the presence of boron hydride, thereby schiff bases is reduced the formation that causes stable secondary amine. Resulting side chain polysaccharide is the oligosaccharide derivative of shitosan, and for example when oligosaccharides is lactose, resulting side chain polysaccharide is the Chitlac that before mentions. Have at derivative in the situation of glycuronic acid of monose or oligosaccharides, reaction is carried out in the presence of suitable activator such as carbodiimides, and causes the formation of acid amides.
The preparation of nano particle
The nano particle that is attached in the polymeric matrix can prepare in the existence of reductive agent or not.
Under first kind of situation, according to following program, by in the aqueous solution of side chain polysaccharide, obtaining nano particle behind the ion of reducing metal with suitable reductive agent, described side chain polysaccharide is made up of the monose of the previous chitosan of mentioning or side chain derivative sugar alcohol or alditol of oligosaccharides: the aqueous solution of these chitosan derivatives prepares under up to the different concns of 2% (w/v), preferably in the scope of 0.05% (w/v), and more preferably be 0.2% (w/v) to 1% (w/v).Then, polysaccharide soln is mixed with metal salt solution, described metal-salt is selected from silver, gold, platinum, palladium, copper, zinc, nickel, preferably is selected from muriate, perchlorate and nitrate (AgNO for example
3, HAuCl
4, CuSO
4, ZnCl
2, NiCl
2), so that obtain the ultimate density to the scope of 20mM,, and more preferably be 1mM more preferably in the scope of 1mM-14mM at 0.1mM.Suitable known reductive agent preferably is selected from xitix, Trisodium Citrate, sodium borohydride and sodium cyanoborohydride, can randomly be added in the solution to obtain the nano particle of metallic state.Reductive agent is added under the concentration from 0.05mM to 10mM, and preferred concentration is 0.5mM.
But find, be different from other polymer systems, also can prepare under the situation of reductive agent not having according to nano-complex of the present invention, because the reductive agent that serves as metal ion itself according to the monose or the oligosaccharides side chain of general formula (I), and allow to form the nano particle that is dispersed in the polymeric matrix.In this case, can obtain metal nanoparticle by simply the solution of chitosan derivatives being mixed with suitable concentration with the salts solution of selected metal.Equally in this case, polysaccharide and metal salt concentrations are reported as previous.
As will be from being easy to significantly by the result that sign obtained who is hereinafter reported to the metal-based nano composite materials of the object of the invention, under all the same situation of all conditions, compare such as for example chitosan with other polysaccharide, the monose of chitosan or oligosaccharide derivative and particularly Chitlac prove astoundingly and are more suitable for forming metal nanoparticle, as the intensity by the UV-visible absorption spectrum and shape with by by the auxiliary spectrophotometry of transmission electron microscope (TEM) research institute proof.These researchs allow to confirm, in the presence of the monose of chitosan or oligosaccharide derivative and particularly Chitlac, metal nanoparticle is compared more even dimensionally and is disperseed better with those metal nanoparticles with unsubstituted Preparation of Chitosan, owing to the interaction with polymer chain prevents its gathering.The TEM microphotograph also allows the nano particle that has highly uniformly less than the 50nm mean sizes is carried out the distribution of sizes analysis.
Also be surprisingly found out that, even the acquisition metal nanoparticle is possible under the situation that does not have external reductive agent.Found a kind of system, wherein, thereby the reducing power that is generally the side chain substituents of polysaccharide by use obtains to be evenly dispersed in the metal nanoparticle in the polymeric matrix under the situation that does not have external reductive agent.This second kind of preparation method also characterizes by UV-visible spectrum and the microscopical means of TEM.Plasma resonance peak (plasmonic peak) and TEM image have been confirmed round-shaped and have been had successful formation less than the metal nanoparticle of 10nm mean sizes.
The approach of this method is particularly advantageous, because the single component of system provides the ability of reduction, dispersion and nano particle stabilization simultaneously.
In addition, other biology sign according to metal-based nano mixture of the present invention shows, nano-complex of the present invention also presents the antimicrobial acivity of bonded metal, and this effect is very valuable for the application purpose in biomedical and field of pharmacology.In fact, metal for example silver, gold, copper, zinc and the use of nickel in the antimicrobial material field is valuable, particularly for both local organization wounds of treatment human and animal.The nano-complex of the object of the invention can use like this or be used in combination with other thinners and/or pharmaceutically acceptable vehicle.Particularly, these materials can with (not-gellifying) of other non--gelations that are selected from the group of forming by dextran, hyaluronic acid, carboxymethyl cellulose, polygalactomannan, neutral or anionic polysaccharide dilution or the spissated aqueous solution in mix, obtain specific viscoelasticity feature, be used for combining with appropriate excipients and/or thinner and prepare composition, said composition has that the nothing that can be used for treating different anatomic department of the Chinese Academy of Sciences position is hindered or the antimicrobial acivity of the infection of injured skin, mucous membrane and epithelium.With other non--gelations as mentioned previously, neutral or anionic these materials of polysaccharide bonded even also can be applicable to have the exploitation of the instrument that is added with medicine such as gauze, bandage, paster (medicated device) of broad-spectrum anti-microbial activity, to be used in the identical top therapeutics field of being mentioned.
In addition, nano-complex according to the present invention can be used as the grafts (or polymer graft thing) (chelating has the metal nanoparticle of antimicrobial acivity) of the polysaccharide on the activating surface that is made by the macromolecular material that is used for the container that pharmacy and field of food use.The advantage of expection can be to replace existing sterilisation program (for example xeothermic), existing sterilisation program sometimes in addition the final sterility (sterility degree) of the empty receptacle before filling go up and produce sizable homogeneity and lack.In addition, apply macromolecular material and can cause obtaining to have the system of the antimicrobial acivity that is applied to biomedical sector.
Metal-based nano composite materials according to the present invention also can be used in the polymer blend with other polysaccharide (for example amylopectin), to obtain to be used for the film of " antimicrobial " packing, particularly in field of food.
Also found the strong enhancing of the raman spectral signal of polysaccharide, this is attributable to the existence of metal nanoparticle, thereby metal nanoparticle has optimum size and distributes to utilize SERS (surface enhanced Raman spectroscopy) effect, and the operation of some molecular biosensors is all based on this SERS effect.In fact, the Raman spectrum of polysaccharide soln shows not detectable peak experimentally, and in the presence of metal nanoparticle, polysaccharide soln (or even more dilution polysaccharide soln) provides strong and Raman spectrum clearly, thereby proof is strengthened by the signal that nano particle (SERS effect) causes.In these are used, metal-based nano mixture according to the present invention can be used as the detector/marker with molecular function, thereby and be considered to be suitable for the application of optics " bio-imaging ", nano particle in the polymeric matrix that forms as the side chain cationic polysaccharide that is trapped in by derivative sugar alcohol or alditol of chitosan can detect by means of TEM microscope, confocal technology and Raman technology.Therefore, metal-based nano particle according to the present invention can be used for developing new transmitter, this new transmitter is used for coming analysing biomolecules and cell by the existence that utilizes specific biochemical signals, described specific biochemical signals is discerned (Donati by the different cell types on the oligosaccharide derivative of chitosan, I. wait the people, Biomaterials 26,2005,987-998).
Use the side chain polysaccharide to form metal nanoparticle according to the present invention and introduced following advantage:
The existence of the amino the on-polymer chain allows the effective coordination of ionic, and allows effective coordination of metal nanoparticle then;
-monose or oligosaccharides side chain have been set up steric hindrance, make them prevent that metal nanoparticle from assembling and guarantee all even in time stable dispersion of metal nanoparticle;
-under all the same situation of all conditions, compare the formation of the side chain derivative of monose chitosan and alditol or sugar alcohol or oligosaccharides (such as for example Chitlac) to nano particle aspect dimensional homogeneity and particles dispersed much effective (proving) as intensity and shape by the plasma resonance band in the UV-visible absorption spectrum under two kinds of situations with chitosan;
-under the situation that does not have other reductive agent, serve as the reduction of metal ion agent according to derivative alditol or sugar alcohol of the side chain of the monose of general formula (I) or oligosaccharides, and allow to form the nano particle that is dispersed in the polymeric matrix;
" autoreduction " ability of the branched oligosaccharides derivative of-chitosan-metal nanoparticle complex compound allows reduction, and some forms the chemical composition of system;
Simple and the result of-preparation has reproducibility;
-since the broad-spectrum anti-microbial activity of nano-complex and the particular optical performance (SERS) in Raman spectrum is used both, nano-complex has widely to be used;
-with the polymer blend of other polysaccharide (for example amylopectin) in possible purposes, to obtain to be used for the film of " antimicrobial " packing, particularly at field of food.
For illustrative purposes, below described preparation some non-limiting examples, wherein carried out physical-chemical sign and bioassay to estimate the antibacterial activity of nano-complex according to nano-complex of the present invention.
Embodiment 1: the derivative of chitosan and lactose (Chitlac) (1a) and the derivative (Chitcell) of chitosan and cellobiose synthesizing (1b)
1a) chitosan (1.5g, degree of acetylation 11%) is dissolved in the 1%w/v acetate buffer solution (55mL) of the methanol solution (55mL) of 110mL and pH 4.5.Add the methanol solution (30mL) of the 60mL that comprises lactose (2.2g) and sodium cyanoborohydride (900mg) and the 1%w/v acetate buffer solution (30mL) of pH 4.5.Mixture was stirred 24 hours, transfer to dialysis tube (molecular weight cut-off 12000Da), and utilize NaCl 0.1M (changing 2 times) and utilize deionized water to carry out dialysis until the specific conductivity that obtains 4 μ S under 4 ℃.At last, solution is filtered and lyophilize on Millipore 0.45 μ m strainer.
1b) chitosan (1.5g, degree of acetylation 11%) is dissolved in the 1%w/v acetate buffer solution (55mL) of the methanol solution (55mL) of 110mL and pH 4.5.Add the methanol solution (30mL) of the 60mL that comprises cellobiose (2.2g) and sodium cyanoborohydride (900mg) and the 1%w/v acetate buffer solution (30mL) of pH 4.5.Mixture was stirred 24 hours, transfer to dialysis tube (molecular weight cut-off 12000Da), and utilize NaCl 0.1M (changing 2 times) and utilize deionized water to carry out dialysis until the specific conductivity that obtains 4 μ S under 4 ℃.At last, solution is filtered and lyophilize on Millipore 0.45 μ m strainer.
Embodiment 2: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.05% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 0.5mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.25mM.
Embodiment 3: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.05% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 4: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: concentration is the Chitlac aqueous solution of 0.1% (w/v), so that obtain the AgNO of 0.5mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.25mM.
Embodiment 5: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.1% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 6: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 0.5mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.25mM.
Embodiment 7: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 8: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 0.5mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.25mM.
Embodiment 9: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 10: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 5mM
3Ultimate density.
Embodiment 11: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 10mM
3Ultimate density.
Embodiment 12: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 14mM
3Ultimate density.
Embodiment 13: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 5mM
3Ultimate density.
Embodiment 14: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 10mM
3Ultimate density.
Embodiment 15: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 14mM
3Ultimate density.
Embodiment 16: do not having to prepare gold nano grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the sodium borohydride reduction metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with tetra chlorauric acid solution, so that obtain the HAuCl of 1mM
4Ultimate density.Then, add sodium borohydride solution so that obtain the ultimate density of 0.3mM.
Embodiment 17: do not having to prepare gold nano grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the sodium borohydride reduction metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with tetra chlorauric acid solution, so that obtain the HAuCl of 1mM
4Ultimate density.Then, add sodium borohydride solution so that obtain the ultimate density of 0.3mM.
Embodiment 18: do not having to prepare gold nano grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with tetra chlorauric acid solution, so that obtain the HAuCl of 1mM
4Ultimate density.
Embodiment 19: prepare copper nano particles in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the sodium borohydride reduction metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with copper-bath, so that obtain the CuSO of 1mM
4Ultimate density.Then, add sodium borohydride solution so that obtain the ultimate density of 0.2mM.
Embodiment 20: do not having to prepare silver nano-grain under the situation of reductive agent in Chitcell (the cellobiose derivative of chitosan)
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitcell reducing metal ion: preparation concentration is the Chitcell aqueous solution of 0.4% (w/v).Then, Chitcell solution is mixed with silver nitrate solution, so that obtain the AgNO of 14mM
3Ultimate density.
Embodiment 21: do not having to prepare silver nano-grain under the situation of reductive agent in Chitgluc (glucose-derivative of chitosan)
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitgluc reducing metal ion: preparation concentration is the moisture Chitgluc solution of 0.4% (w/v).Then, Chitgluc solution is mixed with silver nitrate solution, so that obtain the AgNO of 14mM
3Ultimate density.
Embodiment 22: prepare silver nano-grain in the presence of reductive agent in chitosan
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using chitosan solution: preparation concentration is the moisture chitosan solution of 0.2% (w/v).Then, chitosan solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 23: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle behind the xitix reducing metal ion in using Chitlac solution: preparation concentration is the Chitlac aqueous solution of 1% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 24: prepare silver nano-grain in the presence of reductive agent in Chitlac
According to following program, the xitix reducing metal ion in using Chitlac solution obtained nano particle afterwards: preparation concentration is the Chitlac aqueous solution of 2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.Then, add ascorbic acid solution so that obtain the ultimate density of 0.5mM.
Embodiment 25: do not having to prepare silver nano-grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.2% (w/v).Then, Chitlac solution is mixed with silver nitrate solution, so that obtain the AgNO of 1mM
3Ultimate density.
Embodiment 26: do not having to prepare gold nano grain under the situation of reductive agent in Chitlac
According to following program, obtain to comprise the nano-complex of nano particle after by polysaccharide Chitlac reducing metal ion: preparation concentration is the Chitlac aqueous solution of 0.4% (w/v).Then, Chitlac solution is mixed with tetra chlorauric acid solution, so that obtain the HAuCl of 1mM
4Ultimate density.
Physical-chemical and biological the sign have been reported below according to metal-based nano mixture of the present invention by the nano particle acquisition of the chitosan derivatives with lactose (Chitlac) and different metal and particularly Ag and Au.All oligosaccharide derivatives of the object of the invention have obtained similar result.
Stablizing of nano particle
For the formation of examining nano particle and stable according in the polymeric matrix of the object of the invention thereof, make the nano-complex of embodiment 7 stand transmission electron microscope.As emphasizing among Fig. 1, the existence of multiple functional group (amino, hydroxyl) has promoted to interact with the coordination of metal ion, and the existence of the side chain of monose or oligosaccharides such as the side chain of for example lactose provides effective steric hindrance, so that stop the natural tendency of nanoparticle aggregate.In fact, when adding AgNO
3Solution has been set up Ag during to Chitlac solution
+Coordination between ion and the amino interacts; After reduction, participate in the stabilization of metal nanoparticle near amino lactose side chain.
The UV-visible light characterizes
Prove forming of nano particle by the appearance at the absorption band at the wavelength place that changes according to the metal considered in the UV-visible absorption spectrum; These bands are known with the surface plasma resonance band, and are that collective oscillation by the unbound electron on the nano grain surface causes.For example, under the situation of silver, observe the strong band (Fig. 2 A) that concentrates on about 400nm place; Symmetry and very narrow plasmon band show, have abundant dispersive, be mainly the nano particle of spherical form.Under all the same situation of all conditions, the different performance of two kinds of polysaccharide has been given prominence in the contrast of the absorption spectrum of the nano particle that forms in Chitlac and chitosan.In fact, under the situation of Chitlac, plasmon band is much better than, how symmetrically how, narrow and towards moving than small wavelength, and this shows less and disperses exist (Fig. 2 A) of better particulate.Compare with chitosan, the preferable performance of Chitlac is attributable to be positioned at the physical-chemical performance of the lactose side chain on the polymer chain.System can adjust according to employed concentration, as characterizing (Fig. 2 B) that is proved by the spectrophotometry of carrying out.The plasma resonance band be particularly important and have a diagnostic value.
The metal particle major part that spectrum has further been confirmed to be included in the polymeric matrix all has zero charge; But, can have bunch (Ag for example that has ion characteristic on a small quantity
4 +), as (Fig. 2, A and the B) as indicated in the acromion at about 260nm place in the UV absorption spectrum of system.
The research that changes according to the UV-visible spectrum in concentration and reaction times is allowed to improve in the best preparation method aspect productive rate, reproducibility and in time stable.
Equally, under the situation of gold, detect the strong plasmon band of about 530nm, this strong plasmon band proof is dispersed in the successful formation (Fig. 3) of the gold nano grain among the Chitlac.
The reducing property of Chitlac
Be surprisingly found out that, under suitable condition, even under the situation of the reductive agent that does not have to add, also can form metal nanoparticle; For example, under the situation of silver, Chitlac can reduce Ag
+Ion and do not need the auxiliary of xitix for example.UV-visible spectrum (Fig. 4) and TEM image (Fig. 5) have been confirmed stable and successful formation abundant dispersive metal nanoparticle.Shall also be noted that under suitable condition, can form hydrogel and need not to add other chemical agent based on the Chitlac of no reductive agent and the system of silver nano-grain.In addition, these gels fluff in time and loose and last softening (about 1 week).
Even under the situation of gold, the Chitlac that does not have the reductive agent that adds also allows Au
3+Ion is reduced to metallic state, as being proved by the UV-visible spectrum among Fig. 3 with by the TEM image among Fig. 6, observes the plasma peak of gold in Fig. 3, observes the nano particle with about 50nm mean sizes in Fig. 6.
TEM characterizes and dimension analysis
Utilize transmission electron microscope (TEM), nano particle trace analysis (Nanosight
) and image analysis technology is estimated in the existence of reductive agent and shape, distribution and the size of the nano particle that forms not.The image relevant with gold nano grain (Fig. 6 and 7) shows the existence of the nano particle with about 50nm mean sizes.Even under the situation of copper, clearly observe the successful formation (Fig. 8) of metal nanoparticle.TEM image round-shaped and silver nano-grain that have about 30nm mean sizes has been shown among Fig. 9.
Also utilize UV-visible spectrum and TEM microscope to characterize the preparation method of this no reductive agent.Plasma peak among Fig. 4 and the TEM image among Fig. 5 have been confirmed round-shaped and have been had successful formation less than the metal nanoparticle of 10nm mean sizes.
In order to estimate antimicrobial acivity, test the nano-complex system with different Gram-positive bacteria strains and Gram-negative bacteria strain; Confirmed strong and sterilization effect fast to the experiment of the growth kinetics of bacterial colony and counting experiment.
Anti-microbial activity:
In the presence of the Chitlac that has and do not have metal nanoparticle, on different bacterium bacterial strain (Gram-negative: intestinal bacteria (Escherichia coli), Pseudomonas aeruginosa (Pseudomonas aeruginosa), and Gram-positive: streptococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcus epidermidis)), carry out the growth-inhibiting experiment.Bacterial growth and the growth phase in contrast (Mueller-Hinton meat soup) of discovery in the Chitlac of no nano particle solution worked as; On the contrary, issue in the existence of the Chitlac with metal nanoparticle and to give birth to sizable bacterial growth and suppress,, wherein can find out minimizing according to the bacterial colony quantity of incubation time as what for example under the situation of silver and golden (Figure 10,11 and 12), reported.This process is very fast: even just real effective inhibition of finding bacterial cell after several hrs.
Raman spectrum characterizes
(Renishaw plc, Wotton-under-Edge U.K.) collect the Raman spectrum of the solution of the nano particle that comprises polymer stabilizing to use the inVia Raman system.(West Jordan UT U.S.A.) focuses on the sample for 514.5nm argon laser, LaserPhysics with laser by 20 * object lens (0.4NA).The laser power at sample place is 3mW; For the sample photodegradation that makes induced with laser minimizes, reduce laser power density by making light beam defocus 10%.Exposure duration is the accumulation (accumulation) of 5 10s of every spectrum, promptly composes the exposure duration of 50s altogether for every.
Found the strong enhancing of the raman spectral signal of polysaccharide, this is attributable to the existence of metal nanoparticle.Therefore, metal nanoparticle has optimum size and distribution of sizes to utilize SERS (surface enhanced Raman spectroscopy) effect, and the operation of some molecular biosensors is all based on this SERS effect.In fact, as from Figure 13, inferring, the Raman spectrum of polysaccharide soln shows can not detected peak, and the phase homopolysaccharide in the presence of metal nanoparticle even solution more dilution provides strong and Raman spectrum clearly, thus the enhancing of the signal that proof is caused by nano particle (SERS effect).
The metal-based nano mixture is used to apply the application of methacrylic resin
With the cylindrical sample of methacrylic resin (
H=2mm) immersed down among the 12M HCl 7 hours at 80 ℃; Then, with deionized water (2 * 50mL), 0.1M NaOH (50mL) and water (50mL) rinse sample again.Will be in acid-treated methacrylic resin sample immerses according to the colloidal solution of the metal-based nano mixture of embodiment 9 preparations 24 hours.At last, rinse sample in deionized water, and under stink cupboard dry sample.
Some biological signs of the methacrylic resin that applies have been reported hereinafter.
Antimicrobial efficacy is analyzed
With the cylindrical sample of the methacrylic resin that applies (
H=2mm) be placed as with the streptococcus aureus bacterium and directly contact.The bacterial suspension of 10 μ L is tiled on each sample, and concentration is 10
6CFU/mL (CFU=colony-forming unit), and hatched in the wet environment under 37 ℃ 3 hours.Then, under agitation each sample is immersed among the 2mL PBS, break away from from the surface to allow bacterium.These washingss are diluted in turn and are tiled on the agar gel, with the enumeration after the night incubation of permission under 37 ℃.Enumeration quantity shows that under the situation of the methacrylic resin that scribbles Chitlac-nAg, lip-deep bacterial number has reduced by 98%.
The LDH cytotoxicity analysis
After antibacterial test, carried out further research, purpose is to estimate the nano-complex coating to eukaryotic toxicity.For this target, by with osteoblast-like cells (MG63) and material itself with both directly contact and carry out the LDH cytotoxicity analysis with the liquid nutrient medium (" extract ") that keeps in touch the given time (24 hours) with this material.The material of being tested (in quadruplicate) directly is deposited on the cellular layer.24 and 72h after, collect substratum and carry out LDH according to the agreement of manufacturers and analyze.Institute's results reported shows among Figure 14, after 72 hours, does not have the Cytotoxic evidence relevant with material.
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Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08268821A (en) * | 1995-04-03 | 1996-10-15 | Sangi Co Ltd | Antibacterial agent composition |
| JP3955652B2 (en) * | 1996-10-15 | 2007-08-08 | 株式会社ネーテック | Synthetic aminosaccharide derivatives and method for producing the same |
| JPH10130427A (en) * | 1996-10-25 | 1998-05-19 | Kazuya Abe | Material comprising compound of metal with chitin derivative and chitosan derivative |
| JPH10158305A (en) * | 1996-12-03 | 1998-06-16 | Ichimaru Pharcos Co Ltd | Antimicrobial and antiseptic agent containing chitosan derivative |
| ITPD20060202A1 (en) * | 2006-05-22 | 2007-11-23 | Univ Degli Studi Trieste | POLYMERIC MIXTURES OF ANIONIC AND CATIONIC POLYSACCHARIDES AND THEIR USE |
| ITPD20060203A1 (en) * | 2006-05-22 | 2007-11-23 | Univ Degli Studi Trieste | HYDROGELS OF POLYSACCHARID MIXTURES FOR TISSUE ENGINEERING AND THE VEHICLE OF ACTIVE COMPOUNDS |
| WO2008076339A2 (en) * | 2006-12-15 | 2008-06-26 | Drexel University | Multilayer films |
| US20080147019A1 (en) * | 2006-12-19 | 2008-06-19 | Kimberly-Clark Worldwide, Inc. | Antimicrobial component system containing metallic nanoparticles and chitosan and/or its derivatives |
-
2008
- 2008-07-23 IT ITPD2008A000219A patent/IT1391668B1/en active
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2009
- 2009-07-22 JP JP2011519163A patent/JP2011528694A/en active Pending
- 2009-07-22 CN CN2009801287434A patent/CN102105494A/en active Pending
- 2009-07-22 ES ES09800059.9T patent/ES2662321T3/en active Active
- 2009-07-22 WO PCT/EP2009/059430 patent/WO2010010122A1/en active Application Filing
- 2009-07-22 BR BRPI0916300A patent/BRPI0916300A2/en not_active IP Right Cessation
- 2009-07-22 EP EP09800059.9A patent/EP2310422B1/en not_active Not-in-force
- 2009-07-22 US US13/055,349 patent/US20110129536A1/en not_active Abandoned
- 2009-07-22 CA CA2731390A patent/CA2731390A1/en not_active Abandoned
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| CN102621091A (en) * | 2012-04-01 | 2012-08-01 | 中国科学院宁波材料技术与工程研究所 | Method for quickly detecting copper ions in solution |
| CN102621091B (en) * | 2012-04-01 | 2014-07-30 | 中国科学院宁波材料技术与工程研究所 | Method for quickly detecting copper ions in solution |
| CN108024958A (en) * | 2015-09-24 | 2018-05-11 | 金善实 | Biological nano gold Polysaccharide solution and its manufacture method |
| CN110501325A (en) * | 2019-02-01 | 2019-11-26 | 上海海关动植物与食品检验检疫技术中心 | A rapid Raman detection method for propyl gallate in olive oil and its blended oil |
| CN111840630A (en) * | 2020-07-21 | 2020-10-30 | 华南农业大学 | Bacteriostatic absorbable medical soft tissue suture thread and preparation method and application thereof |
| CN115350283A (en) * | 2022-03-22 | 2022-11-18 | 四川大学 | A carbohydrate functionalized nanoparticle and its preparation method and application |
| CN115350283B (en) * | 2022-03-22 | 2024-01-26 | 四川大学 | A kind of carbohydrate functionalized nanoparticle and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| BRPI0916300A2 (en) | 2018-06-12 |
| WO2010010122A1 (en) | 2010-01-28 |
| ES2662321T3 (en) | 2018-04-06 |
| IT1391668B1 (en) | 2012-01-17 |
| US20110129536A1 (en) | 2011-06-02 |
| CA2731390A1 (en) | 2010-01-28 |
| JP2011528694A (en) | 2011-11-24 |
| ITPD20080219A1 (en) | 2010-01-24 |
| EP2310422B1 (en) | 2017-12-13 |
| EP2310422A1 (en) | 2011-04-20 |
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